Members of the Smad family are conserved transducers of the TGF-beta- related signaling pathway. Mice null for Smad4, the central mediator of the pathway, die around perigastrulation with multiple patterning and proliferation defects. We have developed a novel genotyped-based chemical mutagenesis method capable of generating mutations of genes of interest in mouse Embryonic Stem (ES) cells. We propose to use this method to generate an allelic series of mutations in Smad4 to reveal additional functions that may be masked by the early lethality seen in the gene-targeted null animals. ES cells will be mutagenized with the point mutagen, ESU. Clones carrying Smad4 mutations will be identified by heteroduplex analysis of the RT/PCR products. To facilitate the stock maintenance and genetic analysis of the ENU-induced Smad4 mutations, an inversion or deletion will be engineered in the Smad4 region using the Cre/loxP mediated recombination. ES cells with Smad4 mutations will be injected into blastocyst to generate chimeras. Germline transmitted mutations will be bred to aggregations chimeras will be used to investigate tissue-specific requirement of SMAD4 in development and tumorigenesis. In addition, various mutant Smad4 proteins will e expressed in Xenopus animal cap tissue or a Smad4 null cell line for biochemical analyses of the molecular mechanisms by which the ENU- induced mutations affect the signaling process. Taken together, the generation of an allelic series of smad4 mutation will provide useful genetic and molecular reagents for a more complete assessment of its role in TGF-beta-related signaling.